Fundamental studies of structure-function relationships of the HIV Protease (HIV PR) will be undertaken with the goal of increasing our understanding of this therapeutically important enzyme. Highly optimized total chemical synthesis will be used to prepare both HIV PR as well as a series of synthetic analogues of the enzyme. These analogues will be rationally designed using molecular modeling to probe the important structure-function relationships implicit in the enzyme's proposed mode of action. Throughout this program the fundamental structural and thermodynamic impact of these modifications will be gauged in a number of ways. The complete 2D-NMR assignment of an appropriately isotopically labeled synthetic enzyme will be undertaken and subsequently used as a structural fingerprint by which to routinely compare analogues. Furthermore, it is anticipated that thermodynamic and kinetic measurements will be made on all synthetic enzymes, again these quantities will be compared with the native enzyme. In addition, X-ray crystallography will be performed on selected analogues. The dynamic nature of the NMR experiment is ideally suited to the study of the flap movements which apparently occur upon substrate binding. The proposed role of Water301 in the catalytic mechanism will be evaluated by site- specific replacement of peptide bonds thought to be involved in H-bonding interactions with substrates/inhibitors through this water molecule. The ionization state of the catalytically active Asp25, Asp125 side chains will be directly determined by single-atom labeling with 13C as an NMR reporter group. Rules governing substrate specificity will be deduced, and will be tested by construction of analog PR molecules with space filling and/or charge modifying geometrically-constrained moieties introduced into the P1 and P1' specificity pockets. The fundamental knowledge resulting from these studies of HIV PR will be important for the understanding of related clinically-relevant enzymes, such as the other retroviral proteases, and of cell-encoded aspartyl proteinases such as renin.